MyoD mRNA Research Articles

Allogenic Vertebral Body Adherent Mesenchymal Stromal Cells Promote Muscle Recovery in Diabetic Mouse Model of Limb Ischemia

Chronic limb-threatening ischemia (CLTI) carries a significant risk for amputation, especially in diabetic patients with poor options for revascularization. Phase I trials have demonstrated efficacy of allogeneic mesenchymal stromal cells (MSC) in treating diabetic CLTI. Vertebral bone-adherent mesenchymal stromal cells (vBA-MSC) are derived from vertebral bodies of deceased organ donors, which offer the distinct advantage of providing a 1,000x greater yield compared to that of living donor bone aspiration. This study describes the effects of intramuscular injection of allogenic vBA-MSC in promoting limb perfusion and muscle recovery in a diabetic CLTI mouse model. A CLTI mouse model was created through unilateral ligation of the femoral artery in male polygenic diabetic TALLYHO mice. The treated mice were injected with vBA-MSC into the gracilis muscle of the ischemic limb 7days post ligation. Gastrocnemius or tibialis muscle was assessed postmortem for fibrosis by collagen staining, capillary density via immunohistochemistry, and mRNA by quantitative real-time polymerase chain reaction (PCR). Laser Doppler perfusion imaging and plantar flexion muscle testing (MT) were performed to quantify changes in limb perfusion and muscle function. Compared to vehicle (Veh) control, treated mice demonstrated indicators of muscle recovery, including decreased fibrosis, increased perfusion, muscle torque, and angiogenesis. PCR analysis of muscle obtained 7 and 30 days post vBA-MSC injection showed an upregulation in the expression of MyoD1 (P=0.03) and MyH3 (P=0.008) mRNA, representing muscle regeneration, vascular endothelial growth factor A (VEGF-A) (P=0.002; P=0.004) signifying angiogenesis as well as interleukin (IL-10) (P<0.001), T regulatory cell marker Foxp3 (P=0.04), and M2-biased macrophage marker Mrc1 (CD206) (P=0.02). These findings indicate human allogeneic vBA-MSC ameliorate ischemic muscle damage and rescue muscle function. These results in a murine model will enable further studies to develop potential therapies for diabetic CLTI patients.

ADAR1 Promotes Myogenic Proliferation and Differentiation of Goat Skeletal Muscle Satellite Cells.

As one of the most important economic traits for domestic animal husbandry, skeletal muscle is regulated by an intricate molecular network. Adenosine deaminase acting on RNA (ADAR1) involves various physiological processes and diseases, such as innate immunity and the development of lung adenocarcinoma, breast cancer, gastric cancer, etc. However, its role in skeletal muscle growth requires further clarification. Here, we explored the functions of ADAR1 in the myogenic process of goat skeletal muscle satellite cells (MuSCs). The ADAR1 transcripts were noticeably enriched in goat visceral tissues compared to skeletal muscle. Additionally, its levels in slow oxidative muscles like the psoas major and minor muscles were higher than in the fast oxidative glycolytic and fast glycolytic muscles. Among the two common isoforms from ADAR1, p110 is more abundant than p150. Moreover, overexpressing ADAR1 enhanced the proliferation and myogenic differentiation of MuSCs. The mRNA-seq performed on MuSCs' knockdown of ADAR1 obtained 146 differentially expressed genes (DEGs), 87 upregulated and 59 downregulated. These DEGs were concentrated in muscle development and process pathways, such as the MAPK and cAMP signaling pathways. Furthermore, many DEGs as the key nodes defined by protein-protein interaction networks (PPI), including STAT3, MYH3/8, TGFβ2, and ACTN4, were closely related to the myogenic process. Finally, RNA immunoprecipitation combined with qPCR (RIP-qPCR) showed that ADAR1 binds to PAX7 and MyoD mRNA. This study indicates that ADAR1 promotes the myogenic development of goat MuSCs, which provides a useful scientific reference for further exploring the ADAR1-related regulatory networks underlying mammal skeletal muscle growth.

Vigeo Promotes Myotube Differentiation and Protects Dexamethasone-Induced Skeletal Muscle Atrophy via Regulating the Protein Degradation, AKT/mTOR, and AMPK/Sirt-1/PGC1α Signaling Pathway In Vitro and In Vivo.

Sarcopenia, a condition caused by an imbalance between muscle growth and loss, can severely affect the quality of life of elderly patients with metabolic, inflammatory, and cancer diseases. Vigeo, a nuruk-fermented extract of three plants (Eleutherococcus senticosus Maxim (ESM), Achyranthes japonica (Miq.) Nakai (AJN), and Atractylodes japonica Koidzumi (AJK)) has been reported to have anti-osteoporotic effects. However, evidence of the effects of Vigeo on muscle atrophy is not available. Here, in the in vivo model of dexamethasone (Dex)-induced muscle atrophy, Vigeo treatment significantly reversed Dex-induced decreases in calf muscle volume, gastrocnemius (GA) muscle weight, and histological cross-section area. In addition, in mRNA and protein analyses isolated from GA muscle, we observed that Vigeo significantly protected against Dex-induced mouse muscle atrophy by inhibiting protein degradation regulated by atrogin and MuRF-1. Moreover, we demonstrated that Vigeo significantly promoted C2C12 cell line differentiation, as evidenced by the increased width and length of myotubes, and the increased number of fused myotubes with three or more nuclei. Vigeo alleviated the formation of myotubes compared to the control group. Vigeo also significantly increased the mRNA and protein expression of myosin heavy chain (MyHC), MyoD, and myogenin compared to that in the control. Vigeo treatment significantly reduced the mRNA and protein expression of muscle degradation markers atrogin-1 and muscle RING Finger 1 (MuRF-1) in the C2C12 cell line in vitro. Vigeo also activated the AMP-activated protein kinase (AMPK)/silent information regulator 1 (Sirt-1)/peroxisome proliferator-activated receptor-γ co-activator-1α (PGC1α) mitochondrial biogenesis pathway and the Akt/mTOR protein synthesis signaling pathway in Dex-induced myotube atrophy. These findings suggest that Vigeo may have protective effects against Dex-induced muscle atrophy. Therefore, we propose Vigeo as a supplement or potential therapeutic agent to prevent or treat sarcopenia accompanied by muscle atrophy and degeneration.

Both rapid hyperplasia and hypertrophy promoted skeletal muscle growth in mandarin fish Siniperca chuatsi, compared to big-eye mandarin fish Siniperca kneri

As important cultured fishes in China, mandarin fish, Siniperca chuatsi and big-eye mandarin fish, Siniperca kneri share similar external morphology and genomes, but have significant differences in growth rates. In this study, we used 6 mph (month post hatching) mandarin fish and big-eye mandarin fish to compare differences in skeletal muscle growth over a one-month period, and use transcriptomes to explore the reasons for the growth differences. The results showed that the different value in number of muscle fibers (2.72 times), density (2.56 times), area (1.95 times) and diameter (2.73 times) of mandarin fish are significantly higher than that of big-eye mandarin fish (P<0.05). Hypertrophy (Contribution rate: 97.9%) is the dominant factor in big-eye mandarin fish, and both hypertrophy and hyperplasia (Contribution rate: 2.1%) were significantly different from mandarin fish (P<0.05). Transcriptome results showed that differential genes were enriched in proliferation of satellite cells, protein synthesis and degradation. Further analysis of the transcriptome results showed that the number of Pax7-positive cells per unit area of mandarin fish was 2.3 fold higher that of big-eye mandarin fish (P<0.05). Myod, pax3, pax7, and myf5 mRNA expression of mandarin fish were significantly higher than those of big-eye mandarin fish(P<0.05). In muscle fiber fusion, mef2, myog, and myomaker mRNA expression of mandarin fish were significantly higher than that of big-eye mandarin fish (P < 0.05). In protein deposits, the results showed that the muscle fiber area of mandarin fish was 2.85 times larger than that of big-eye mandarin fish by comparing the muscle fiber area with the same number of nuclei. Polr2a, mtorc1, 4e-bp1, s6, e1, murf, foxo3, atg5, beclin, and atg14 mRNA expression of mandarin fish were significantly higher than that of big-eye mandarin fish (P < 0.05), while eef2, eif2α, gcn2, eif2b, capn and cast of mandarin fish was significantly lower than that of big-eye mandarin fish (P < 0.05). This work verified that the skeletal muscle growth rate of mandarin fish is faster than that of big-eye mandarin fish. The greater number and higher activity of satellite cells of mandarin fish increased hyperplasia efficiency, and the faster fusion rate and protein precipitation rate increased hypertrophy efficiency. Both hyperplasia and hypertrophy contribute to faster skeletal muscle growth of mandarin fish than that of big-eye mandarin fish.

Effects of Ulmus macrocarpa Extract and Catechin 7-O-β-D-apiofuranoside on Muscle Loss and Muscle Atrophy in C2C12 Murine Skeletal Muscle Cells.

Muscle atrophy is known to be one of the symptoms leading to sarcopenia, which significantly impacts the quality of life, mortality, and morbidity. Therefore, the development of therapeutics for muscle atrophy is essential. This study focuses on addressing muscle loss and atrophy using Ulmus macrocarpa extract and its marker compound, catechin 7-O-β-D-apiofuranoside, by investigating their effects on biomarkers associated with muscle cell apoptosis. Additionally, protein and gene expression in a muscle atrophy model were examined using Western blotting and RT-PCR. Ulmus macrocarpa has been used as food or medicine due to its safety, including its roots, barks, and fruit. Catechin 7-O-β-D apiofuranoside is an indicator substance of plants of the Ulmus genus and has been reported to have various effects such as antioxidant and anti-inflammatory effects. The experimental results demonstrated that catechin glycoside and Ulmus macrocarpa extract decreased the expression of the muscle-degradation-related proteins Atrogin-1 and Muscle RING-Finger protein-1 (MuRF1) while increasing the expression of the muscle-synthesis-related proteins Myoblast determination (MyoD) and Myogenin. Gene expression confirmation experiments validated a decrease in the expression of Atrogin and MuRF1 mRNA and an increase in the expression of MyoD and Myogenin mRNA. Furthermore, an examination of muscle protein expression associated with the protein kinase B (Akt)/forkhead box O (FoxO) signaling pathway confirmed a decrease in the expression of FoxO, a regulator of muscle protein degradation. These results confirm the potential of Ulmus macrocarpa extract to inhibit muscle apoptosis, prevent muscle decomposition, and promote the development of functional materials for muscle synthesis, health-functional foods, and natural-product-derived medicines.

Impaired myoblast differentiation and muscle IGF-1 receptor signaling pathway activation after N-glycosylation inhibition.

The role of N-glycosylation in the myogenic process remains poorly understood. Here, we evaluated the impact of N-glycosylation inhibition by Tunicamycin (TUN) or by phosphomannomutase 2 (PMM2) gene knockdown, which encodes an enzyme essential for catalyzing an early step of the N-glycosylation pathway, on C2C12 myoblast differentiation. The effect of chronic treatment with TUN on tibialis anterior (TA) and extensor digitorum longus (EDL) muscles of WT and MLC/mIgf-1 transgenic mice, which overexpress muscle Igf-1Ea mRNA isoform, was also investigated. TUN-treated and PMM2 knockdown C2C12 cells showed reduced ConA, PHA-L, and AAL lectin binding and increased ER-stress-related gene expression (Chop and Hspa5 mRNAs and s/uXbp1 ratio) compared to controls. Myogenic markers (MyoD, myogenin, and Mrf4 mRNAs and MF20 protein) and myotube formation were reduced in both TUN-treated and PMM2 knockdown C2C12 cells. Body and TA weight of WT and MLC/mIgf-1 mice were not modified by TUN treatment, while lectin binding slightly decreased in the TA muscle of WT (ConA and AAL) and MLC/mIgf-1 (ConA) mice. The ER-stress-related gene expression did not change in the TA muscle of WT and MLC/mIgf-1 mice after TUN treatment. TUN treatment decreased myogenin mRNA and increased atrogen-1 mRNA, particularly in the TA muscle of WT mice. Finally, the IGF-1 production and IGF1R signaling pathways activation were reduced due to N-glycosylation inhibition in TA and EDL muscles. Decreased IGF1R expression was found in TUN-treated C2C12 myoblasts which was associated with lower IGF-1-induced IGF1R, AKT, and ERK1/2 phosphorylation compared to CTR cells. Chronic TUN-challenge models can help to elucidate the molecular mechanisms through which diseases associated with aberrant N-glycosylation, such as Congenital Disorders of Glycosylation (CDG), affect muscle and other tissue functions.

Effects of three short-chain fatty acids on growth, intestinal microbiota composition, and ammonia tolerance of juvenile yellow catfish Pelteobagrus fulvidraco

The objective of this research was to examine how the growth, ammonia tolerance, and intestinal microbiota composition of juvenile yellow catfish are affected by dietary supplementation of isobutyric acid (IBA), isovaleric acid (IVA), and 2-methylbutyric acid (2-MBA). During the 8-week trial, four groups were fed the basal diet (Control), and the basal diet supplementation with 0.25% IBA, IVA and 2-MBA, respectively. The research shows that the yellow catfish in the IBA and 2-MBA groups had a higher weight gain rate and specific growth rate than the control group, but feed conversion ratio has the opposite trend. Dietary supplementation of IBA and 2-MBA can significantly improve digestive enzymes activities (α-amylase, lipase, and trypsin) in intestinal tract and blood health (serum albumin increased, alanine transaminase and aspartate transaminase decreased). The up-regulation of IGF-1, Myod, and MyoG gene mRNA expression levels occurred significantly in the muscle tissue of the IBA, IVA, and 2-MBA groups, but IL 1 and IL8 genes in intestinal tract were significantly down-regulated. Supplementation of IBA, IVA and 2-MBA significantly increased the relative abundance of Candidatus Arthromitus, while decreased the relative abundance of Achromobacter, most notably in IBA group. After 96-h ammonia stress, cumulative mortality in IBA group was the lowest. Supplementing the diet of yellow catfish with IBA or 2-MBA can enhance their growth, digestive enzyme activity, and blood health status. Additionally, IBA can improve ammonia tolerance, which may be related to the increase abundance of Candidatus Arthromitus in intestine.

Effects of glycyrrhiza polysaccharides on growth performance, meat quality, serum parameters and growth/meat quality-related gene expression in broilers.

With growing restrictions on the use of antibiotics in animal feed, plant extracts are increasingly favored as natural feed additive sources. Glycyrrhiza polysaccharide (GP), known for its multifaceted biological benefits including growth promotion, immune enhancement, and antioxidative properties, has been the focus of recent studies. Yet, the effects and mechanisms of GP on broiler growth and meat quality remain to be fully elucidated. This study aimed to investigate the effects of GP on growth, serum biochemistry, meat quality, and gene expression in broilers. The broilers were divided into five groups, each consisting of five replicates with six birds. These groups were supplemented with 0, 500, 1,000, 1,500, and 2,000 mg/kg of GP in their basal diets, respectively, for a period of 42 days. The results indicated that from day 22 to day 42, and throughout the entire experimental period from day 1 to day 42, the groups receiving 1,000 and 1,500 mg/kg of GP showed a significant reduction in the feed-to-gain ratio (F:G) compared to the control group. On day 42, an increase in serum growth hormone (GH) levels was shown in groups supplemented with 1,000 mg/kg GP or higher, along with a significant linear increase in insulin-like growth factor-1 (IGF-1) concentration. Additionally, significant upregulation of GH and IGF-1 mRNA expression levels was noted in the 1,000 and 1,500 mg/kg GP groups. Furthermore, GP significantly elevated serum concentrations of alkaline phosphatase (AKP) and globulin (GLB) while reducing blood urea nitrogen (BUN) levels. In terms of meat quality, the 1,500 and 2,000 mg/kg GP groups significantly increased fiber density in pectoral muscles and reduced thiobarbituric acid (TBA) content. GP also significantly decreased cooking loss rate in both pectoral and leg muscles and the drip loss rate in leg muscles. It increased levels of linoleic acid and oleic acid, while decreasing concentrations of stearic acid, myristic acid, and docosahexaenoic acid. Finally, the study demonstrated that the 1,500 mg/kg GP group significantly enhanced the expression of myogenin (MyoG) and myogenic differentiation (MyoD) mRNA in leg muscles. Overall, the study determined that the optimal dosage of GP in broiler feed is 1,500 mg/kg.

Cloning, phylogenetic and expression analysis of two MyoDs in yellowtail kingfish (Seriola lalandi)

Yellowtail kingfish (Seriola lalandi) is a pelagic piscivore distributed circumglobally. Owing to its great market value, the growth mechanism of S. lalandi, including muscle development and growth, is a hot research topic. The myoblast determination protein (MyoD) gene has been shown to play an important role in formation of myoblasts and the function of somites in fish. The open reading frame (ORF) sequences of MyoD1 and MyoD2 in S. lalandi encoded 298 and 263 amino acids possessing three common characteristic domains, respectively, containing a myogenic basic domain, a bHLH domain, and a ser-rich region (helix III). S. lalandi MyoDs shared the highest identity with the MyoDs of S. dumerili. MyoDs are highly expressed in white muscle (P < 0.05) in S. lalandi. The expression level of MyoD1 mRNA was higher than that of MyoD2 mRNA during embryonic and early developmental stages, indicating that the two MyoD isoforms may have different roles in muscle formation. Moreover, the mRNA expression of MyoDs in the brain, pituitary, liver and muscle of endocrine growth axis were analyzed in the various sizes and ages stages. The expression levels of MyoDs in the different sizes and ages of S. lalandi showed that expression of both these genes was particularly high in 400-g fish and 2-year-old fish (P < 0.05). Moreover, the increases in the mRNA expression and plasma levels of growth hormone (GH) and insulin-like growth factor (IGF-I) were accompanied by an increase in mRNA expression of MyoDs, indicating the roles of GH and IGF-I in muscle development and growth of S. lalandi. Overall, the expression profiles of genes associated with muscle development are the first step taken towards deciphering fast growth mechanism in this important Seriola fish.

Characteristics of bovine muscle satellite cell from different breeds for efficient production of cultured meat.

The purpose of this study was comparing in vitro performances of three breeds of donor satellite cells for cultured meat and selecting the optimal donor and providing insight into the selection of donors for cultured meat production. Cattle muscle satellite cells were isolated from the muscle tissue of Hanwoo, Holstein, and Jeju black cattle, and then sorted by fluorescence activated cell sorting (FACS). Regarding proliferation of satellite cells, all three breeds showed similar trends. The myogenic potential, based on PAX7 and MYOD mRNA expression levels, was similar or significantly higher for Holstein than other breeds. When the area, width, and fusion index of the myotube were calculated through immunofluorescence staining of myosin, it was expressed upward for Holstein in all experiments except myotube area at passage 8. In addition, it was confirmed that Holstein's muscle satellite cells showed an upward expression in the amount of gene and protein expression related to myogenic. In the case of gene expression of MYOG, DES, and MYH4 known to play a key role in differentiation into muscles, it was confirmed that Holstein's muscle satellite cells expressed higher levels. CAV3, IGF1 and TNNT1, which contribute to hypertrophy and differentiation of muscle cells, showed high expression in Holstein. Our results suggest using cells from Holstein cattle can increase the efficiency of cultured meat production, compared to Hanwoo and Jeju breeds, because the cells exhibit superior differentiation behavior which would lead to greater yields during the maturation phase of bioprocessing.

The novel RNA-RNA activation of H19 on MyoD transcripts promoting myogenic differentiation of goat muscle satellite cells

The elaborate interplay of coding and noncoding factors governs muscle growth and development. Here, we reported a mutual activation between long noncoding RNA (lncRNA) H19 and MyoD (myogenic determination gene number 1) in the muscle process. We successfully cloned the two isoforms of goat H19, which were significantly enriched and positively correlated with MyoD transcripts in skeletal muscles or differentiating muscle satellite cells (MuSCs). To systematically screen genes altered by H19, we performed RNA-seq using cDNA libraries of differentiating H19-deficiency MuSCs and consequently anchored MyoD as the critical genes in mediating H19 function. Intriguingly, some transcripts of MyoD and H19 overlapped in the cytoplasm, which was dramatically damaged when the core complementary nucleotides were mutated. Meanwhile, MyoD RNA successfully pulled down H19 in MS2-RIP experiments. Furthermore, HuR could bind both H19 and MyoD transcripts, while H19 or its truncated mutants successfully stabilized MyoD mRNA, with or without HuR deficiency. In turn, novel functional MyoD protein-binding sites were identified in the promoter and exons of the H19 gene. Our results suggest that MyoD activates H19 transcriptionally, and RNA-RNA hybridization is critical for H19-promoted MyoD expression, which extends our knowledge of the hierarchy of regulatory networks in muscle growth.

Detection of multiple biomarkers associated with satellite cell fate in the contused skeletal muscle of rats for wound age estimation.

From the perspective of forensic wound age estimation, experiments related to skeletal muscle regeneration after injury have rarely been reported. Here, we examined the time-dependent expression patterns of multiple biomarkers associated with satellite cell fate, including the transcription factor paired box 7 (Pax7), myoblast determination protein (MyoD), myogenin, and insulin-like growth factor (IGF-1), using immunohistochemistry, western blotting, and quantitative real-time PCR in contused skeletal muscle. An animal model of skeletal muscle contusion was established in 30 Sprague-Dawley male rats, and another five rats were employed as non-contused controls. Morphometrically, the data obtained from the numbers of Pax7 + , MyoD + , and myogenin + cells were highly correlated with the wound age. Pax7, MyoD, myogenin, and IGF-1 expression patterns were upregulated after injury at both the mRNA and protein levels. Pax7, MyoD, and myogenin protein expression levels confirmed the results of the morphometrical analysis. Additionally, the relative quantity of IGF-1 protein > 0.92 suggested a wound age of 3 to 7days. The relative quantity of Pax7 mRNA > 2.44 also suggested a wound age of 3 to 7days. Relative quantities of Myod1, Myog, and Igf1 mRNA expression > 2.78, > 7.80, or > 3.13, respectively, indicated a wound age of approximately 3days. In conclusion, the expression levels of Pax7, MyoD, myogenin, and IGF-1 were upregulated in a time-dependent manner during skeletal muscle wound healing, suggesting the potential for using them as candidate biomarkers for wound age estimation in skeletal muscle.

Aerobic conditioning alters the satellite cell and ribosome response to acute eccentric contractions in young men and women.

Satellite cells (SCs) and ribosomes are key determinants of the skeletal muscle adaptive response. Both are thought to increase acutely after resistance exercise and chronically with resistance training. However, the acute SC and ribosome exercise response with prior aerobic conditioning is unknown. Fourteen young men and women underwent 6 wk of single-legged aerobic conditioning followed by an acute bout of 300 eccentric contractions on each leg. Muscle biopsies were taken from the vastus lateralis of the aerobically conditioned (AC) and the control (CTL) legs before (Pre), 24 (24 h), and 48 (48 h) h post-contractions. Pre-eccentric contractions, 45S pre-rRNA and 5.8S internal transcribed spacer (ITS) expression were lower in the AC leg compared with the CTL leg. SC content (PAX7+ cells/100 fibers) in type I and mixed fibers showed a main effect of condition, where values were greater in the AC leg compared with the CTL. A main effect of condition for Pax7 and MyoD1 mRNA expression was observed where expression was greater in the AC leg compared with the CTL. AC had greater RNA concentration and mRNA expression of Ubf and Tif-1a compared with CTL. Only the AC leg increased (Pre-24h) 45S pre-rRNA, 5.8S ITS, and 28S ITS following eccentric contractions. We discovered that aerobic conditioning increased type-I SC abundance and the acute increase in ribosome content following eccentric contractions.

Synergistic Effect of Hydrogen and 5-Aza on Myogenic Differentiation through the p38 MAPK Signaling Pathway in Adipose-Derived Mesenchymal Stem Cells.

This study aims to clarify the systems underlying regulation and regulatory roles of hydrogen combined with 5-Aza in the myogenic differentiation of adipose mesenchymal stem cells (ADSCs). In this study, ADSCs acted as an in vitro myogenic differentiating mode. First, the Alamar blue Staining and mitochondrial tracer technique were used to verify whether hydrogen combined with 5-Aza could promote cell proliferation. In addition, this study assessed myogenic differentiating markers (e.g., Myogenin, Mhc and Myod protein expressions) based on the Western blotting assay, analysis on cellular morphological characteristics (e.g., Myotube number, length, diameter and maturation index), RT-PCR (Myod, Myogenin and Mhc mRNA expression) and Immunofluorescence analysis (Desmin, Myosin and β-actin protein expression). Finally, to verify the mechanism of myogenic differentiation of hydrogen-bound 5-Aza, we performed bioinformatics analysis and Western blot to detect the expression of p-P38 protein. Hydrogen combined with 5-Aza significantly enhanced the proliferation and myogenic differentiation of ADSCs in vitro by increasing the number of single-cell mitochondria and upregulating the expression of myogenic biomarkers such as Myod, Mhc and myotube formation. The expressions of p-P38 was up-regulated by hydrogen combined with 5-Aza. The differentiating ability was suppressed when the cells were cultivated in combination with SB203580 (p38 MAPK signal pathway inhibitor). Hydrogen alleviates the cytotoxicity of 5-Aza and synergistically promotes the myogenic differentiation capacity of adipose stem cells via the p38 MAPK pathway. Thus, the mentioned results present insights into myogenic differentiation and are likely to generate one potential alternative strategy for skeletal muscle related diseases.

Dietary protein levels changed the hardness of muscle by acting on muscle fiber growth and the metabolism of collagen in sub-adult grass carp (Ctenopharyngodon idella)

BackgroundNutrient regulation has been proven to be an effective way to improve the flesh quality in fish. As a necessary nutrient for fish growth, protein accounts for the highest proportion in the fish diet and is expensive. Although our team found that the effect of protein on the muscle hardness of grass carp was probably related to an increased collagen content, the mechanism for this effect has not been deeply explored. Moreover, few studies have explored the protein requirements of sub-adult grass crap (Ctenopharyngodon idella). Therefore, the effects of different dietary protein levels on the growth performance, nutritional value, muscle hardness, muscle fiber growth, collagen metabolism and related molecule expression in grass carp were investigated.MethodsA total of 450 healthy grass carp (721.16 ± 1.98 g) were selected and assigned randomly to six experimental groups with three replicates each (n = 25/replicate), and were fed six diets with 15.91%, 19.39%, 22.10%, 25.59%, 28.53% and 31.42% protein for 60 d.ResultsAppropriate levels of dietary protein increased the feed intake, percentage weight gain, specific growth rate, body composition, unsaturated fatty acid content in muscle, partial free amino acid content in muscle, and muscle hardness of grass carp. These protein levels also increased the muscle fiber density, the frequency of new muscle fibers, the contents of collagen and IGF-1, and the enzyme activities of prolyl 4-hydroxylases and lysyloxidase, and decreased the activity of matrix metalloproteinase-2. At the molecular level, the optimal dietary protein increased collagen type I α1 (Colα1), Colα2, PI3K, Akt, S6K1, La ribonucleoprotein domain family member 6a (LARP6a), TGF-β1, Smad2, Smad4, Smad3, tissue inhibitor of metalloproteinase-2, MyoD, Myf5, MyoG and MyHC relative mRNA levels. The levels of the myostatin-1 and myostatin-2 genes were downregulated, and the protein expression levels of p-Smad2, Smad2, Smad4, p-Akt, Akt, LARP6 and Smad3 were increased.ConclusionsThe appropriate levels of dietary protein promoted the growth of sub-adult grass carp and improved muscle hardness by promoting the growth of muscle fibers, improving collagen synthesis and depressing collagen degradation. In addition, the dietary protein requirements of sub-adult grass carp were 26.21% and 24.85% according to the quadratic regression analysis of growth performance (SGR) and the muscle hardness (collagen content), respectively.

Exon skipping in genes encoding lineage-defining myogenic transcription factors in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a childhood sarcoma composed of myoblast-like cells, which suggests a defect in terminal skeletal muscle differentiation. To explore potential defects in the differentiation program, we searched for mRNA splicing variants in genes encoding transcription factors driving skeletal muscle lineage commitment and differentiation. We studied two RMS cases and identified altered splicing resulting in “skipping” the second of three exons in MYOD1. RNA-seq data from 42 tumors and additional RMS cell lines revealed exon 2 skipping in both MYOD1 and MYF5 but not in MYF6 or MYOG. Complementary molecular analysis of MYOD1 mRNA found evidence for exon skipping in five additional RMS cases. Functional studies showed that so-called MYODΔEx2 protein failed to robustly induce muscle-specific genes, and its ectopic expression conferred a selective advantage in cultured fibroblasts and an RMS xenograft. In summary, we present previously unrecognized exon skipping within MYOD1 and MYF5 in RMS, and we propose that alternative splicing can represent a mechanism to alter the function of these two transcription factors in RMS.

Modification of beneficial fatty acid composition and physicochemical qualities in the muscle of sub-adult grass carp (Ctenopharyngodon idella): The role of lipids

Flesh quality, including beneficial fatty acid composition and physicochemical qualities, is the most important factor for many consumers. The actions and mechanisms of promotion of polyunsaturated fatty acid (PUFA) contents, muscle hardness, and related signalling pathways by lipids were investigated in sub-adult grass carp. In this experiment, 450 healthy grass carp (initial body weight 756.57 ± 1.28 g) were selected and randomly divided into 6 treatments, and fish in each group were fed diets with different lipid levels (0.79, 1.58, 2.72, 4.06, 5.16, and 6.33%) for 60 days. The results showed that compared with the control group (0.79% lipid diet), the optimal lipid levels in the diet increased the percentage of weight gain (PWG), feed intake (FI) and flesh rate (P < 0.05), indicating that lipids improved the growth performance of sub-adult grass carp. The optimal levels of lipids also increased muscle PUFA content, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (P < 0.05), which demonstrated that lipids improved beneficial fatty acid composition in the muscle. Meanwhile, optimal lipid levels increased muscle shear force, collagen content and myofibre density (P < 0.05), which indicated that lipids improved muscle physicochemical qualities. We further explored the mechanism of lipid-increased muscle PUFA content and muscle hardness in fish. We observed that the increase in muscle PUFA content by optimal lipid levels may be related to the promotion of LC-PUFA biosynthesis and catabolism signalling pathways (LXRα/SREBP-1, Sp1, and PPARα) in fish. Meanwhile, the enhancement of muscle hardness by optimal levels of lipids may be associated with the increase in collagen content as well as the improvement of the muscle histological characteristics (diameter and density) in fish. The increase in the muscle collagen content may be related to lipids motivating collagen synthesis pathways including the TGF-β1/Smads pathway at the transcriptional level and the PI3K/Akt/LARP6 signalling pathway at the initial stage of translation. In addition, the increase in muscle fibre density may be attributed to lipid-upregulated MRFs (MyoD, MyoG and Myf5) mRNA levels and decreased myostatin (MSTN1 and MSTN2) mRNA levels (P < 0.05), which were involved in myofibre development. Collectively, lipid-improved flesh quality, including beneficial muscle fatty acid composition and physicochemical qualities, is connected with the activation of related signalling pathways and regulatory factors. Moreover, based on PWG and muscle shear force, the dietary lipid requirements for sub-adult grass carp (756–1459 g) were 4.01% and 3.65% of the diet, respectively.

Effects of PGC-1α overexpression on the myogenic response during skeletal muscle regeneration

The ability of skeletal muscle to regenerate from injury is crucial for locomotion, metabolic health, and quality of life. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1A) is a transcriptional coactivator required for mitochondrial biogenesis. Increased mitochondrial biogenesis is associated with improved muscle cell differentiation, however PGC1A's role in skeletal muscle regeneration following damage requires further investigation. The purpose of this study was to investigate the role of skeletal muscle-specific PGC1A overexpression during regeneration following damage. 22 C57BL/6J (WT) and 26 PGC1A muscle transgenic (A1) mice were injected with either phosphate-buffered saline (PBS, uninjured control) or Bupivacaine (MAR, injured) into their tibialis anterior (TA) muscle to induce skeletal muscle damage. TA muscles were extracted 3- or 28-days post-injury and analyzed for markers of regenerative myogenesis and protein turnover. Pgc1a mRNA was ∼10–20 fold greater in A1 mice. Markers of protein synthesis, AKT and 4EBP1, displayed decreases in A1 mice compared to WT at both timepoints indicating a decreased protein synthetic response. Myod mRNA was ∼75% lower compared to WT 3 days post-injection. WT mice exhibited decreased cross-sectional area of the TA muscle at 28 days post-injection with bupivacaine compared to all other groups. PGC1A overexpression modifies the myogenic response during regeneration.

Enhanced Muscle Fibers of Epinephelus coioides by Myostatin Autologous Nucleic Acid Vaccine.

Epinephelus coioides is a fish species with high economic value due to its delicious meat, high protein content, and rich fatty acid nutrition. It has become a high-economic fish in southern parts of China and some other Southeast Asian countries. In this study, the myostatin nucleic acid vaccine was constructed and used to immunize E. coioides. The results from body length and weight measurements indicated the myostatin nucleic acid vaccine promoted E. coioides growth performance by increasing muscle fiber size. The results from RT-qPCR analysis showed that myostatin nucleic acid vaccine upregulated the expression of myod, myog and p21 mRNA, downregulated the expression of smad3 and mrf4 mRNA. This preliminary study is the first report that explored the role of myostatin in E. coioides and showed positive effects of autologous nucleic acid vaccine on the muscle growth of E. coioides. Further experiments with increased numbers of animals and different doses are needed for its application to E. coiodes aquaculture production.

Dietary enzymatic rice protein and enzymatic fish paste affect the growth, muscle development and quality traits of juvenile channel catfish (Ictalurus punctatus)

Fishmeal serves as the major protein for aquafeeds, whose limitations are observed owing to its high price and dependence on imports. Enzymatic rice protein (RP) and enzymatic fish paste (FP) were evaluated as potential alternatives to fishmeal in industrial aquafeeds. A basal fishmeal diet (10% fishmeal) was used as a control (FM), then three diets were formulated with fishmeal replaced by RP in amounts of 2.5% (RP2.5), 5.0% (RP5.0) and 7.5% (RP7.5). To further reduce dietary fishmeal, two diets based on the RP5.0 (5.0% fishmeal) were formulated, in which FP replaced fishmeal at levels of 2.5% (FP2.5) and 5.0% (FP5.0). The six diets were fed thrice daily to channel catfish (Ictalurus punctatus, initial mean weight 6.50 g) for eight weeks to evaluate growth and muscle quality. The weight gain rate (WGR) of fish was highest in RP2.5 group and lowest in RP7.5 group (P < 0.05). No significant differences in WGR were observed among the RP5.0, FP2.5, FP5.0 and FM groups (P > 0.05). Further, the RP7.5 diet significantly reduced total protein content of muscle than FM diet. Conversely, dietary FP (FP2.5, FP5.0) increased total protein and crude lipid content, and significantly improved muscle textures (hardness, gumminess, and chewiness), coupled with a significant increase in transverse section area of myofibers (AMF). Real-time qPCR showed that FP supplement significantly up-regulated muscle myod mRNA and down-regulated mstn mRNA, thereby regulating the myofiber development, which in turn affected muscle texture. Conversely, RP7.5 diet significantly down-regulated myod mRNA and resulted in a decrease in density of myofibers (DMF). Further, RP2.5 and RP5.0 diets significantly increased catalase (CAT) activity of muscle. FP supplement significantly up-regulated muscle gsh-px4b mRNA and increased the activities of CAT and glutathione peroxidase (GSH-PX). In summary, replacing 2.5% fishmeal with RP could improve growth performance, while growth was reduced when it reached to 7.5%, possibly by inhibiting muscle development. Notably, fishmeal could be completely replaced by a mixture of 5.0% FP and 5.0% RP, contributing to maintaining growth as well as promoting muscle development and antioxidant properties.